HRMAS: 1H-15N HSQC of fractionally deuterated (mostly at Calpha) KcsA

Citation

A.G. Palmer III, J. Cavanagh, P.E. Wright & M. Rance, J. Magn. Reson. 93, 151-170 (1991) https://www.sciencedirect.com/science/article/pii/002223649190036S L.E. Kay, P. Keifer & T. Saarinen, J. Am. Chem. Soc. 114, 10663-5 (1992) (https://pubs.acs.org/doi/abs/10.1021/ja00052a088](https://pubs.acs.org/doi/abs/10.1021/ja00052a088) J. Schleucher, M. Schwendinger, M. Sattler, P. Schmidt, O. Schedletzky, S.J. Glaser, O.W. Sorensen & C. Griesinger, J. Biomol. NMR 4, 301-306 (1994) [https://www.ncbi.nlm.nih.gov/pubmed/8019138]https://www.ncbi.nlm.nih.gov/pubmed/8019138) S. Grzesiek & A. Bax, J. Am. Chem. Soc. 115, 12593-12594 (1993) https://pubs.acs.org/doi/abs/10.1021/ja00079a052

Protocol

a. Increasing spinning frequency above 8kHz doesn't bring any improvements in most cases. b. There are two water populations due to the liposome sample. They can be both efficiently suppressed by the pulse sequence. c. Use EA only d. SMSQ10.100 have much better performance than SINE.100 e. You can adjust p28 trim pulse length between 500us and 1000us to optimize water suppression vs protein signal loss. f. P11 shaped 1H pulse is not in EDPROSOL table. Calculate it using stdisp. g. Use halfG half Gauss shape for EA pulse sequences. Use Integrate shape analyze integr3 option in STDISP to calculate power level. h. optimize SP1 in GS mode to minimize water signal. i. Optimize GPZ3 coherence selection gradient pulse +/- 1%

Samples

The experiments were performed with a precipitated 2F-2H, U-13C/15N KcsA, LPR =1, 9:1 DOPE-DOPS D2O citrate buffer pH 3.5

Processing and analysis

Pulse Sequence and Parameter Set